The present invention relates to the synthesis and use of synthetic peptides and proteins to induce an immune response in experimental animals. More particularly, the present invention relates to a polyamide resin, a method of making that polyamide resin, a method of inducing an immune response in an experimental animal using a conjugate of a peptide or protein synthesized on that resin, and the the use of the resin for immunodiagnostic purposes.
Solid phase peptide synthesis is a valuable tool for investigating the structure and mechanism of action of proteins. Most such synthetic methods involve the use of a cross-linked polystyrene based resin as the solid phase to which the peptide is anchored during assembly, usually through a linker molecule. Assembly is accomplished by a repetitive cycle of adding a protected amino acid to the solid phase, selectively removing (deprotecting) a protective group on that amino acid, and adding additional suitably protected amino acids (for a review, see Merrifield, R.B., "Solid-phase Peptide Synthesis", 32 Adv. Enzymology 221 (1969)).
Although cross-linked, polystyrene based resins are most commonly used as supports in solid phase peptide synthesis, their relatively hydrophobic character in comparison to the polar organic media required to solubilize reactants can be problematic in peptide chain assembly. Such media may freely solvate the growing peptide, yet incompletely swell the polystyrene matrix. Within the polymer lattice, impaired diffusion of reagents and steric hindrance can contribute to lowered efficiency during coupling cycles, which, on a repeated basis, lowers final yields appreciably. During the early stages of assembly, when the resin to peptide mass ratio is high and the physical properties of the support dominate, this lowered efficiency is particularly acute.
Those shortcomings led to the development of a cross-linked, polydimethylacrylamide based support which is highly polar in character and is freely permeated by the requisite solvents for peptide synthesis. Atherton, E., D.L.J. Clive and R.C. Sheppard, "Polyamide Supports For Polypeptide Synthesis", 97 J. Amer. Chem. Soc. 6584 (1975); Arshady, R., E. Atherton, M.J. Gait, K. Lee and R.C. Sheppard, "Easily Prepared Polar Support For Solid Phase Peptide And Oligonucleotide Synthesis". 1979 J.C.S. Chem. Comm. 425 (1979). The polyamide resin, as the amino methyl derivative, can accommodate synthetic schemes incorporating alternate protection strategies through selection of the appropriate linker molecule, which links the C-terminal residue to the support. The peptide or protein thus synthesized, which will be referred to throughout the present disclosure as a "protide", can be used in a number of investigative applications.
Of particular interest to the present invention is the use of the protide as an immunogen. It has previously been demonstrated that synthetic peptides analogous to sequences contained in viral encoded proteins have proven useful for identification of native antigen determinants associated with such proteins. Several laboratories have reported studies on the antigenic activity of various HBsAg synthetic peptides. Dreesman, G.R., et al., 295 Nature 158 (1982); Lerner, R.A., et al. 78 Proc. Natl. Acad. Sci. USA 3403 (1981); Prince, A.M., et al., 79 Proc. Natl. Acad. Sci. USA 579 (1982). The induction of an antibody response to HBsAg, using such peptides, proved to be relatively weak, but could be enhanced through coupling of peptides to a carrier protein prior to immunization. Lerner, et al., supra; Sanchez, Y., et al., 18 Intervirology 209 (1982). Because the prediction of potential antigenic determinants of immunogenic proteins based on primary sequences analysis is not exact, the identification of putative epitopes through trial and error can be laborious. A method which involves the delineation of native antigenic sequences with synthetic peptides which does not require purification of the synthetic peptide and coupling of the peptide to carrier proteins offers significant advantages. It is, therefore, an object of the present invention to provide a method of preparing a polyamide resin upon which a protide can be synthesized using solid phase synthetic methods which can be injected into an experimental animal to induce an immunogenic response without separation of the protide from the resin.
It is another object of the present invention to provide a polyamide resin for solid phase protide synthesis, and a conjugate of that polyamide resin and synthesized protide, which can be injected into an experimental animal to induce an immunogenic response.
It is another object of the present invention to provide a polyamide resin-protide conjugate for use in in vitro immunological assays.
Another object of the present invention is to provide a method of preparing a polyamide resin for solid phase protide synthesis comprising cross-linking a dimethylacrylamide monomer with a molecule containing a functional group by free radical co-polymerization in an aqueous solution which has been emulsified by a detergent when added to an organic solvent medium.
Another object of the present invention is to provide a method of inducing an immunogenic response in an experimental animal with a synthetic peptide or protein comprising preparing a polyamide resin, synthesizing a peptide or protein on that polyamide resin, and immunizing an experimental animal with the polyamide resin-synthetic peptide or synthetic protein conjugate.
Another object of the present invention is to provide an assay for detection of proteins such as antigens and antibodies using the polyamide resin-protide conjugate of the present invention.
Another object of the present invention is to provide a method of inducing an immunogenic response in an experimental animal using the polyamide resin-protide conjugate of the present invention.
Another object of the present invention is to provide a polyamide resin for solid phase protide synthesis which does not require the separation of the protide from the resin and the subsequent purification of the protide before the use of that protide to, for instance, induce an immunogenic response.
Another object of the present invention is to provide a polyamide resin for solid phase protide synthesis which is particularly useful in the mapping of the antigenic determinants of a protein as a result of the elimination of the steps of separation of the protide from the resin and the subsequent purification of the protide before the use of the protide in a binding assay.
These and other objects and advantages of the present invention will be clear to those skilled in the art from the following description.